Bioelectronic Measurement of Target Engagement to a Membrane-Bound Transporter.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable.

Dynamic Reorganization and Correlation among Lipid Raft Components.

Lipid rafts are widely believed to be an essential organizational motif in cell membranes. However, direct evidence for interactions among lipid and/or protein components believed to be associated with rafts is quite limited owing, in part, to the small size and intrinsically dynamic interactions that lead to raft formation. Here, we exploit the single negative charge on the monosialoganglioside GM1, commonly associated with rafts, to create a gradient of GM1 in response to an electric field applied parallel to a patterned supported lipid bilayer.

Colocalization of the ganglioside G(M1) and cholesterol detected by secondary ion mass spectrometry.

The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remain a major challenge. The concept of lipid rafts is widely invoked; however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of secondary ion mass spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano- and microscale assemblies in a canonical lipid raft composition of lipids.

Vesicles tethered to microbubbles by hybridized DNA oligonucleotides: flow cytometry analysis of this new drug delivery vehicle design.

Hybridization of complementary lipid-linked DNA oligonucleotides was used to tether small unilamellar vesicles (SUVs) to the lipid monolayer shells of air-microbubbles, a new attachment design for a drug delivery vehicle to be used in tandem with ultrasound imaging. Flow cytometry was used, and a novel analysis was developed, based upon light scattering and fluorescence intensity, to quantify the fraction of microbubbles of chosen size-ranges with oligonucleotide-tethered fluorescently labeled SUVs. Fluorescence microscopy was used to verify that our methodology results in successful high-density SUV tethering to a similar fraction of the microbubbles when compared to the flow cytometry statistics.

Microbubbles coated with disaturated lipids and DSPE-PEG2000: phase behavior, collapse transitions, and permeability.

Saturated diacyl (disaturated) phosphatidylcholine (PC) mixed with the lipopolymer distearoylphosphatidylethanolamine (DSPE)-polyethyleneglycol molecular weight 2000 (PEG2000) self-assemble as a monolayer at the air-water interface of air-in-water micrometer-scale bubbles (microbubbles), similar to coatings (shells) on leading medical ultrasound contrast agents (UCAs). This system is characterized here to study the impact of the DSPE-PEG2000 species and PC chain-length on the monolayer coating phase behavior, collapse, shedding, and air transport resistance and microbubble dissolution rate and surface contour. Using fluorescence microscopy of dissolving microbubbles, we found that film microstructure and collapse behavior for all chain lengths (n = 14-20) was indicative of primarily condensed phase monolayers, unlike similar coatings containing polyethyleneglycol 40 stearate (PEG40S) that are either expanded phase or coexisting expanded-condensed phase monolayers.

Long-term stability by lipid coating monodisperse microbubbles formed by a flow-focusing device.

In this letter, the long-term stabilization of monodisperse microbubbles produced by flow focusing is demonstrated using lipid encapsulation. Fluorescence microscopy, high-speed camera imaging, and particle size analysis were used to investigate the roles of lipid phase behavior, dissolution, Ostwald ripening, and coalescence in the stability of microbubbles formed by flow focusing. It was found that these behaviors were controlled through compositional changes with respect to lipid, emulsifier, and viscosity agents.

Simultaneous detection of double-stranded RNA-induced protein kinase and its specific mRNA by single cell analysis.

Fluorescent in situ hybridization was combined with flow cytometry to detect the expression of the double-stranded-RNA-induced protein kinase (PKR) in single cells. Labeled anti-sense oligonucleotide was used to target the specific mRNA while the protein was targeted with an antibody. It was demonstrated that the PKR-mRNA signal could be protected through a lengthy immunostaining procedure.